In some immuno-histochemical detection systems a target antigen present in the histology sample is detected by a double antibody system. Initially the sample is incubated with a primary targeting antibody that is specific for the target antigen. Detection of antigen-antibody complexes containing the primary antibody and formed during the first incubation is accomplished by incubation with a second detecting antibody that binds to a region of the constant domain in the primary antibody; the second antibody is labeled. The result of the second incubation is, in the presence of the target antigen, a complex of antigen and layers of antibodies that contain the label.
A problem arises in these staining processes when both the targeting antibody (that binds to the target antigen to be detected) and the specimen in which the antigen is to be detected are from the same or related species. Under these conditions, due to the homology of antigenic determinants on the targeting antibody and tissue, the detecting antibody reacts with endogenous antigenic determinants in the tissue causing significant background staining, thus reducing the usefulness of the assay.
Prior attempts to reduce non-specific background in homologous staining systems have used Fab' fragments to block the recognition of the tissue antigenic determinants. When a human anti-tumor antibody was used to stain human tissue sections, Fab' fragments of anti-human antibody were incubated with the tissue section prior to incubation with the targeting primary antibody and secondary detecting antibody. Nielsen et al. (1987) used Fab' rabbit anti-human IgG and Fab' rabbit anti-human IgM as blockers and reported successful blocking with the IgM system, but the background was not abolished in the IgG system. Nielsen, B. et al. (1987), Hybridoma 6:103. Foulds et al. attempted to improve the Fab' blocking process with a prolonged washing step of 20 hours. Foulds, S. and Eremin, O (1988), Hybridoma 9:91.